Project Summary/Abstract The kinetochore connects chromosomes to spindle microtubules to mediate chromosome segregation at cell division. It holds on to microtubules and regulates cell cycle progression, and must do so robustly and accurately, even as microtubules grow, shrink and pull. Errors can lead to disease and birth defects. Our long term research goal is to define the basic physical design principles of the mammalian kinetochore, a machine with dozens of part types, each with hundreds of copies. While we know nearly all its molecules, and in many cases their individual properties, how they collectively give rise to the kinetochore?s emergent decision-making and mechanics remains poorly understood. To close this gap, we need approaches for controlling kinetochore composition and exerting forces on kinetochores inside cells, which we recently developed. Here, we ask how the basic systems engineering features of the mammalian kinetochore ? its detection sensitivity (Aim 1), mechanical robustness (Aim 2), and dynamic feedback (Aim 3) ? emerge from its complex ensemble of parts. In Aim 1, we determine how the mammalian kinetochore integrates microtubule attachment information to regulate anaphase entry. Our recent work suggests that the binding of just a few microtubules are sufficient to satisfy the spindle assembly checkpoint. We test hypotheses on where this sensitivity comes from: how the kinetochore integrates attachment information, sets its detection threshold, and detects early attachment intermediates. We do so by quantitatively tuning kinetochore composition. In Aim 2, we define the molecular basis of the mammalian kinetochore?s robust grip on microtubules. Our recent work suggests that kinetochore proteins are specialized for binding growing or shrinking microtubules. Using laser ablation and cell confinement, we test hypotheses for the molecular basis of robust grip ? specialization in binding activities (Ndc80, Ska1, Astrin/SKAP) and redundancy in component numbers ? and whether they confer robustness to high opposing force. In Aim 3, we determine how mechanical force regulates the dynamics of kinetochore- microtubules to move chromosomes and ensure correct attachments. Based on our preliminary findings, we test the hypothesis that kinetochore-fibers can intrinsically set their baseline dynamics and lifetime, and that these are tuned locally by force at both kinetochores and poles. To test this hypothesis, we will exert local force on kinetochore-fibers using microneedles in mammalian cells of different molecular backgrounds. Establishing the kinetochore?s physical design principles will build on existing structural and biochemical information to provide a framework for understanding its physical function, and will connect diverse kinetochore molecular architectures and physical functions across evolution. Looking forward, it may also allow us to better control and target physical functions for therapy. More broadly, this work will serve as a platform for understanding how other cellular structures, such as cell adhesions and cell-cell junctions, can accurately integrate information and be mechanically robust despite their dynamics.